Extended Data Fig. 10: Clock^C195S mutation was significantly associated with ageing characteristics.

a-b, Representative motifs enriched among gained rhythmic genes of YW (a) and OP2-NM groups (b) relative to OW group in RNA-seq data. c, Representative motifs enriched in ageing-related upregulated DEPs in ATAC-seq data from OP1-NM livers. d, Representative immunoblot showing the reduced thiol of cysteine (Cys-SH) of CLOCK proteins in the liver over a 24-hour period for YW, OW, OP1-ND, and OD groups throughout the diurnal cycle (n = 3 per group; internal control: YW ZT6). e, Density plot showing the density of CLOCK occupancy across the genome in OW (blue) and OP1-NM group (red). f, Bar plot indicating numbers of the upregulated (red) or downregulated (blue) CLOCK occupancy in OP1-NM group across genomic features in CUT&Tag data (n = 3 per group). g, Dot plot showing the enriched GO pathways of downregulated CLOCK-binding regions in OP1-NM group. h, H₂O₂ levels throughout diurnal cycle in liver of Clock^C195S and Clock^WT mice at 3–4 months (Clock^WT: ZT0/6/12/18 n = 6, ZT24 n = 5; Clock^C195S: ZT0 n = 4, ZT6 n = 6, ZT12/18/24 n = 5). i, PCA plot showing individual samples in Clock^C195S and Clock^WT groups in ATAC-seq (left) and RNA-seq (right). j, Violin plot comparing the open accessibilities of genes whose rhythmicity was reprogrammed by ageing (left) and random genes with the same number (right) in Clock^C195S and Clock^WT group (3–4 months; n = 3 per group). The expression for each gene is represented by log2 count per million (CPM) of sequencing reads. k-l qPCR analysis of Ucp1 (k) and Il6 (l) expression in brown fat (BF) from indicated groups (YW n = 7, OW/OP2-NM n = 8, OP1-NM n = 9), with young mice (YW, 3–6 months) and old mice (OW, OP1-NM and OP2-NM, 18-21 months) following the same experimental protocol as described in Fig. 2a. Data are presented as (d, h, k, l) means ± SEM and (j) median values +/- SEM. The center line indicates the median; the box represents the interquartile range (25th-75th percentiles); whiskers extend to 1.5-fold of the interquartile range. P values were calculated using (a-c) hypergeometric test, (g) one-sided hypergeometric test with B–H correction, (h) JTK_Cycle algorithm, (j) Wilcoxon test, and (k-l) Kruskal–Wallis test followed by Dunn’s multiple comparisons hoc test.

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